The overall focus of our laboratory is the study of the 70-kDa heat shock proteins and their role in both normal cellular processes and heat shock. First, we are investigating one of the only defined functions of a 70-kDa heat shock protein--the ability of the 70-kDa uncoating (UC) ATPase isolated from bovine brain to remove clathrin from clathrin coated vesicles in an ATP dependent reaction. Our results show that when the UC ATPase with bound ATP is mixed with coated vesicles, there is an initial burst of uncoating followed by slow steady-state uncoating. Based on these data we have proposed a model where the UC ATPase rapidly removes a stoichiometric amount of clathrin, while ATP is hydrolyzed at the active site. Slow release of ADP and Pi from the resulting enzyme.clathrin.ADP.Pi complex then limits the rate at which further uncoating can occur. This model predicts that, during the steady-state uncoating reaction, a ternary complex composed of the UC ATPase, clathrin, and nucleotide will be present. In support of this model, using FPLC, we have been able to isolate a long lived enzyme-clathrin-ADP.Pi complex following the initial burst of uncoating. We have also been able to show that a similar complex forms when free clathrin is mixed with the UC ATPase. The amount of complex which forms depends on whether equilibrium or steady-state conditions prevail. In addition to these binding studies, we have cloned the bovine brain UC ATPase and plan to express it in a yeast strain which lacks the endogenous genes which produce proteins which we have shown have similar activity to the bovine brain UC ATPase.